Development of a simple, rapid multiplex PCR tool kit by using the 16S rRNA gene for the identification of faecal and non-faecal coliforms in drinking water

Abstract

A multiplex method for the detection of faecal and non-faecal coliforms in drinking water was developed using three primers from the V2, V3 and V9 variable regions of the 16S rRNA gene. 194F, 474F and 1436R are the three primers designed for specific amplification of the V2, V3 and V9 hyper variable regions of the 16S rRNA gene. Multiplex polymerase chain reaction (PCR) allows for differentiation of total coliforms from faecal coliforms by specific amplicons: 1,285 bp of amplicon is specific for six non-faecal coliform genera and 1,009 bp of amplicon is specific for faecal coliform ie. E. coli. If drinking water was contaminated with both faecal and non-faecal coliforms then two amplicons of 1,285 bp and 1,009 bp by combination of the three primers are observed. A multiplex PCR assay based on the 16S rRNA gene should be a beneficial tool kit for the rapid identification of total coliforms in a large number of water samples compared with traditional methods. Results can be acquired within 3 hrs compared with the classic most probable number (MPN) method (3–4 days). This assay will be useful in diversification and detection of seven genera of total coliforms by using variable regions of 16S rRNA. HIGHLIGHTS Rapid method to differentiate both faecal and non-faecal coliforms in water.; Uses V2, V3 and V9 variable regions of 16S rDNA for primer designing and amplified product with the 1,009 bp band specific for E. coli and 1,285 bp band specific for 7 genera of total coliform, whereas two bands in the gel indicates faecal and non-faecal coliforms.; Accurate results when compared to other conventional methods.;

Keywords

• 16s rrna
• coliform
• e. coli
• multiplex pcr
• primer design